Int.J.Curr.Microbiol.App.

Sci (2015) 4(10): 23-32

ISSN: 2319-7706 Volume 4 Number 10 (2015) pp. 23-32

http://www.ijcmas.com

Original Research Article

Isolation of Bioluminescent Bacteria and Their Application in
Toxicity Testing of Chromium in Water

Varsha Shivaji Kulkarni* and Bhagyashree Shashikant Kulkarni

Department of Microbiology, Sinhgad College of Science, Ambegaon (Bk), Pune-411046, India

*Corresponding author

ABSTRACT

Nature has its own beauty and phenomenon called bioluminescence proves it.
Marine fishes like Japanese Threadfin brin (Nemipterus japonicas), Indian Mackerel
(Restrelliger kanagurta) and Anchovy (Engraulis japonicas) were chosen as source
of bioluminescent bacteria. Scales, gills and eye portion along with gut region of
Indian Mackerel were suspended overnight in sterile sea salt saline and from this
loopful suspension were streak inoculated on sterile Photobacterium agar, LA, Sea
water agar and Boss Agar. By subsequent subculturing two luminescent isolates
Keywords named as C1, C2 were obtaining on LA plate from the gut portion of the Indian
Mackerel. They were used for further study. Morphological and biochemical
Biolumine- characterization along with observation of PHB granules under light microscope
scence, suggests that the isolates may belong to Genus Vibrio. To reconfirm these results the
Luciferase, isolates were inoculated on TCBS agar which is the highly specific media for the
Auto- Vibrio identification and confirmation; which confirmed that the isolate C1 may be
induction, late sucrose fermenter Vibrio species. This isolate showed green coloured colonies
Pollutant after 36hrs of incubation. The TSI slant examination stated that the organisms were
chromium, Gram negative enteric pathogen that ferments glucose, lactose and sucrose by acid
Toxicity production without accumulation of gas. After examination of growth pattern of both
testing isolates; C1 and C2 were used for the checking toxicity of the chemical pollutant
cultivation chromium in water. According to the United States Environmental Protection
Agency for primary drinking water the standard Maximum Contaminant Level is 0.1
mg/L for total chromium. Standard protocols were used to determine total chromium
level in water. Performance of the TVC of the both isolates lead to the results that
the pollutant chromium inhibits the growth of bioluminescent bacteria and also
affects the luminescence intensity. This whole study provides eco friendly and cost
effective examination of pollutant chromium. The method can also be used to
measure other chemicals, metal salts like mercuric chloride, zinc chloride and lead
acetate etc.

Introduction

Bioluminescence a Greek word ( Bios for This type of chemiluminescent phenomenon

living and Latin word lumen for light ) have been seen in many types of organisms
which literally means a living light. (bacteria, fungi, fishes, squid,

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dinoflagellates) and have always gained an bacteria and algae gaining the attention for a
attention of scientists because of their toxicity evaluation as a model organisms
beauty, physiology, biochemistry, genetics (Roda et al., 2004; Boynton, 2009). As well
and ease of detection (Lee 2008; known; general metabolism, operon
Vedprakash et al., 2014). In late 19th century expression, bacterial growth, cultural
Raphael Dubois experimentally extracted condition affects the luminescence activity;
the two key components, enzyme from which one can articulate that the
luciferase and luciferine of bioluminescent organisms are very much
bioluminescence reaction which are able to sensitive to any change (e.g. physical,
generate light (Makemson, 1986; Lee, chemical, radiological, etc.) in environment.
2008). Many bacteria regulate their set of Bioluminescent bacterial tests have been
lux genes by the mechanism of quorum successfully used previously in determining
sensing or autoinduction in which the toxicity of aquatic samples, sediments,
autoinducers (AI) evoke the characteristic and soils (Lapota et al., 1998; Kurvet et al.,
response from the cell (Rees et al., 1998; 2005).
Nyholm et al., 2004; Walker et al., 2006). In
bioluminescence after achieving specific Materials and Methods

Anchovy (local name: Mandeli); Indian

threshold value (conc. of AI 10 cell mL ¹ in Mackerel (local name: Bangda); and
Threadfin brin (local name: Raja Rani) these
three fresh marine water fishes were selected
culture media) it triggers the synthesis of as a source of bioluminescent bacteria and
enzyme luciferase and other enzymes were obtained from fish market near Alpna
involved in luminescence (Cao and Meighen Talkies, Camp, Pune. They were kept on ice
and soon after brought to the laboratory for
further processes.
1989; Rees et al., 1998 Walker et al., 2006;
Following fish parts were used as a source
for luminescent bacteria (Table 1) (Walker
et al., 2006, web links).
Berleman, 2009 . This sensing (AI level) Table.1 Fish parts used for luminescent
bacteria
help to ensure that the luminescence
Eye Gill Scales Fin Gut
production is enough high to cause an portion region
impact in environment and making the Anchovy - Used Used - -
process cost effective (Nealson et al., 1970; Mackerel - Used Used - Used
Visick et al., 2000). As the production of the Fim Used Used Used Dorsal -
luminose enzyme increases ultimately light bream fin
generation also increases (Nealson et al.,
1970; Cao and Meighen, 1989; Berleman, These different parts were suspended in
2009). sterile sea saline that is 3.5g of sea salt in
100ml distilled water for 24hrs and from this
Due to the issues raised by animal rights and loopful suspension was streaked for
human rights, the use of microbes like isolation (Web link) on sterile

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 Int.J.Curr.Microbiol.App.Sci (2015) 4(10): 23-32

Photobacterium agar (PBA) casein enzyme selective for Vibrios and differential due to
hydrolysate 5g/L, yeast extract 2g/L, presence of sucrose and the dyes. TCBS
ammonium chloride 0.3g/L, magnesium agar (proteose peptone 10g/L, yeast extract
sulfate 0.3g/L, ferric chloride 0.01g/L, 5g/L, sodium thiosuphate 10g/L, sodium
calcium chloride 1g/L, potassium phosphate citrate 10g/L, oxagall 8g/L, sucrose 20g/L,
(monobasic) 3g/L, sodium glycerophosphate NaCl 10g/L, ferric citrate 1g/L,
23.5g/L, sodium chloride 30g/L, agar 15g/L bromothymol blue 0.40g/L, thymol blue
(web link), sea water agar Part A: yeast 0.40g/L, agar 15g/L, pH 8.6±0.2 at 25 C,
extract 5g/L, peptic digests of animal tissue formula adjusted, standardized) is highly
5g/L, beef extract 3g/L, agar 15g/L, Part B: selective, meets the nutritional requirements
sodium chloride 24g/L, potassium chloride of Vibrio spp., and allows Vibrios to
0.7g/L, magnesium chloride. 6H O 5.3g/L, compete with intestinal flora (Engebrech et
magnesium sulfate. 7H O.7g/L, calcium al., 1983; Musa et al., 2008; Ransanga et al.,
chloride0.1g/L, pH 7.3 +/ 0.2 (web link), 2012; Kannahi and Shivshankari, 2014)
Luminus agar (LA) sodium chloride 10g/L, TCBS Agar is recommended by APHA
yeast extract 5g/L, peptone10g/L, (American Public Health Association) for
agar15g/L, pH 7.3 (web link) and the selective isolation of V. cholerae and V.
bioluminescent agar (Boss agar) sodium parahaemolyticus. Enrichment in alkaline
chloride30g/L, glycerol1g/L, peptone10g/L, peptone water (M618), followed by isolation
meat extract3g/L, pH 7.3 plates (web link). on TCBS Agar is routinely used for isolation
Plates were incubated at RT for 48 72hrs of V. cholerae. Vibrios are natural
and observed for bioluminescence (web inhabitants of sea water (Kumar, 2010). It is
link). possible that a few sucrose-positive Proteus
strains can grow to form yellow colonies.
Identification of isolated bioluminescent Vibrio parahaemolyticus is a sucrose non-
species fermenting organism and produces blue-
green colonies, as does Vibrio vulnificus
Two isolates were obtained by subsequent (Engebrech et al., 1983; Musa et al., 2008;
sub-culturing on sterile LA plates. The Orengo et al., 2010; Ransanga et al., 2012;
isolated were named as C1 and C2. After Kannahi and Shivshankari, 2014).
morphological colony characterization,
Gram staining, motility and Poly- -hydroxy- Triple sugar iron agar (TSI)
butyrate staining (by Sudan Black B
method) were performed (Bergey s manual TSI is recommended in the identification of
9th edition, web link). Bergey s Manual of gram-negative enteric pathogens in routine
determinative Bacteriology 9th edition was examination of faeces. This medium
referred and accordingly biochemical tests indicates the ability of an organism to
like IMViC, amylase, oxidase, nitrate ferment lactose, sucrose and glucose with
reduction were performed and fermentation formation of acid and gas and also its ability
of sugars were also checked with 12 to produce hydrogen sulfide gas (Musa et
different sugars (Table 2). al., 2008; Kumar, 2010; Ransanga et al.,
2012; Kannahi and Shivshankari, 2014)
Growth on Thiosulfate Citrate Bile (Table 3 and 4).
Sucrose Agar (TCBS)
Growth curve
TCBS Agar is both differential and highly

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 Int.J.Curr.Microbiol.App.Sci (2015) 4(10): 23-32

Loopful of 24 hr old culture of isolate C1 Orange Indicator solution, Conc.

and isolate C2 were inoculated in 50 ml Ammonium hydroxide, Potassium
Sterile Luminous Broth(LB) and were kept permanganate solution: Dissolve 4 g
overnight till absorbance reached 0.1 at 600 KMnO in 100ml water, Diphenylcarbazide
nm. After 0.1 absorbance was reached, solution: Dissolve 250 mg in 50ml acetone
growth curve experiment was performed by and Store in brown bottle (APHA, 2012).
taking absorbance at 600nm at 30 minutes Protocol: (APHA, 2012; Tchobanoglous et
interval time period (Eberhard, 1972; al., 2004). Pipette a portion of digested
Engebrech et al., 1983; Cao and Meighen, sample into 125 ml conical flasks, Add few
1989; Kumar, 2010; Gokhale et al., 2012) drops of methyl orange indicator and then
(Graph 1 and 2). add conc. Ammonium Hydroxide until
solution just begins to turn yellow, Add
Application of bioluminescent bacteria in 1+11 ml of sulphuric acid until it turns
toxicity testing of chromium acidic+1ml sulphuric acid in excess, Adjust
the volume to 40ml, Add porcelain pieces
The bacterial bioluminescence test (BBT) is and boil, Add 2 drops of potassium
metabolic inhibition test that uses a permanganate solution to give a dark red
standardized suspension of luminescent colour, if it fades add more potassium
bacteria as test organisms under permanganate, Boil for 2mins, Add 1ml
standardized conditions. This test method sodium azide solution, If red colour doesn t
provides a rapid, reliable and convenient fade completely after boiling for
means of determining the toxicity of waste approximately 30 seconds, add another 1ml
material. We have performed this test for sodium azide solution and continue boiling
checking out the chromium (heavy metal) for 1 min, after color has faded completely,
toxicity on one of our bioluminescent then cool, Add 0.25 ml ortho-phosphoric
bacterium. 0.1 mg of potassium dichromate acid and using 0.2N sulphuric acid adjust pH
was added in 100ml distilled water (APHA, to 1 for yellow colour development and take
2012, Tchobanoglous et al., 2004). O.D. at 540 nm. A 24 hour old culture was
Following protocol was used to convert total inoculated in sterile Luminous Broth to
chromium to the hexavalent state chromium adjust the O.D of 0.1 at 600nm absorbance.
by oxidation with potassium permanganate. C2 was used for this testing. 4ml of such
The oxidation process may not provide total absorbance adjusted broth culture was
conversion of all chromium species to Cr . exposed 1ml of different metal salt solution
For total chromium determination, acid- that is potassium dichromate (K Cr O ) at
digestion of the sample was done. The different concentration up to 60 minutes.
hexavalent chromium is determined Samples were withdrawn at different time
colorimeterically by reaction with intervals of 10, 20, 30, 40, 50, 60 minutes
diphenycarbazide in acid solution. A red- and serially diluted up to 10 . Dilutions
violet colures complex of unknown 10 ³and 10 were plated out on sterile LA
composition is produced. The reaction is
plates. Sample of unexposed culture was
very sensitive, the molar absorbstivity based
plated out as control (Eberhard, 1972;
on chromium being about 40,000 L /g/cm
Engebrech et al., 1983, Cao and Meighen,
at540nm (APHA, 2012; Tchobanoglous et
1989; Kumar, 2010, Gokhale et al., 2012;
al., 2004).
APHA, 2012; Tchobanoglous et al., 2004).
Chemicals Required: Conc. Nitric Acid,
Conc. Ortho-phosphoric acid, Methyl Results and Discussion

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 Int.J.Curr.Microbiol.App.Sci (2015) 4(10): 23-32

Orengo et al., 2010; Ransanga et al., 2012;

Observing plates in dark revealed that the Kannahi and Shivshankari, 2014) (Table 2).
colonies obtain from the gills and scales of To reconfirm the present results both of the
Fim Brim inoculums and gut of Indian isolates were streak inoculated on TCBS
Mackerel inoculums were glowing agar which is specific for Vibrio
beautifully also in presence of other identification (Musa et al., 2008; Kumar,
bacterial population. After subsequent sub- 2010, Ransanga et al., 2012). Our finding
culturing on LA it had been observed that indicates that the isolate C2 may be late
the colonies were obtain from the gut flora sucrose fermenters as it showed green
of Indian Mackerel were glowing with more coloured colonies after 36hrs which was
intensity than the colonies obtain from gills showing yellow colonies after 24hrs of
and scales of Fim Brim. And because of this incubation while C1 showed green colonies
these colonies were chosen for the after 24hrs of incubation. Hydrogen sulfide
experimental purpose (Engebrech et al., gas production was absent for both C1 and
1983; Musa et al., 2008; Orengo et al., C2 (Musa et al., 2008; Ransanga et al.,
2010; Ransanga et al., 2012; Kannahi and 2012). These results confirm that both the
Shivshankari, 2014; web link). Two isolates were from Genus Vibrio (Engebrech
different bioluminescent isolates were et al., 1983; Musa et al., 2008; Orengo et
observed on the LA plates and were al., 2010; Kumar, 2010, Ransanga et al.,
examine for further studies (Photo 1). 2012; Kannahi and Shishankari, 2014).
Microscopic examination of these two Inoculation of C1 and C2 on TSI slants
isolates states that the circular, creamy white identify that organisms were Gram negative
colonies having convex elevation with enteric pathogens (Musa et al., 2008;
smooth (C1) and Mucoid (C2) consistency Ransanga et al., 2012) C1 fermented the
were Gram negative motile short rods lactose or sucrose or both the sugars in
(Bergey s Manual, 9th edition) Both of the absence of H S in acidic condition while C2
isolates C1 and C2 found to be poly- - fermented glucose by producing acid in
hyrdoxy butarate granules non producers; as absence of gas (Engebrech et al., 1983;
no accumulation of PHB were seen in the Musa et al., 2008; Orengo et al., 2010;
cells after staining by Sudan Black B Kumar, 2010, Ransanga et al., 2012;
method (Bergey s Manual, 9th edition; Kannahi and shishankari, 2014) (Table 3
Ransanga et al., 2012). By referring and 4). The important growth curve
Bergey s manual of determinative experiment of both the isolates indicates that
bacteriology (9th edition), biochemical test there was rapid increase in absorbance
were performed on both the isolates. Both of showed by C1 and C2 isolates were showing
the isolates were able to produce enzyme steady increase in cell number. This
gelatinase, catalase and cytocrome oxidase information gave an idea regarding when
and showed negative results for IMViC and cells are going to be in log phase. In log
enzyme amylase production. All 12 chosen phase bioluminescent activity is more
sugars were fermented by C1 and C2 with prominent (Eberhard, 1972; Cao and
acid production in absence of gas (Bergey s Meighen, 1989; Gokhale et al., 2012) and all
Manual, 9th edition; Musa et al., 2008; because of this reason this experiment was
Ransanga et al., 2012). These results the key for the whole toxicity experiment
indicates that the both isolates belong to the (Eberhard, 1972; Engebrech et al., 1983;
genus Vibrio (Bergey s Manual, 9th edition; Cao and Meighen, 1989; Kumar, 2010;
Engebrech et al., 1983; Musa et al., 2008; Gokhale et al., 2012) (Graph 1 and 2).

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Heavy metal toxicity testing

10 ³ and 10 dilution. Comparison with
These bioluminescent bacteria were used to
check toxicity of heavy metal chromium.
Chromium may exist in water supplies in control plates which was free from
both the hexavalent and trivalent form chromium ions showed presence of
although trivalent form rarely occurs in 396CFU/ml ×10 but it was found to be
potable water. The U.S. EPA (United States gradually decreasing as found in table 5.
Environmental Protection Agency) primary These results conclude that the presence of
drinking water standard MCL (Maximum chromium affect the bioluminescent
Contaminant Levels) is 0.1 mg/L for total bacterial growth. So the use of this
chromium (APHA, 2012; Tchobanoglous, bioluminescent bacteria for the toxicity
2004). So serially diluted sample was testing was successfully accomplished with
checked for the total viable count between easy detection (APHA, 2012;
Tchobanoglous, 2004).

Table.2 Characteristic and biochemical test results

Isolate C-1 C-2

Morphology
Gram character -/S -/S
Motility + +
Consistency Smooth Mucoid
PHB Production - -
Luminescence +/blue colour +/green colour
Fermentation of sugar
Arabinose A A
Fructose A A
Galactose A A
Glucose A A
Lactose A A
Maltose A A
Manitol A A
Mannose A A
Rhamnose A A
Ribose A A
Sucrose A A
Xylose A A
Biochemical Tests
Indole test - -
Methyl Red test - -
Voges-Prosker test - -
Citrate - -
Nitrate Reduction test - -
Catalase test + +
Amylase test - -

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Gelatinase test + +
Oxidase test + +
Key -/S= Gram negative short rods, A= acid production, no gas production was observed

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 Int.J.Curr.Microbiol.App.Sci (2015) 4(10): 23-32

Table.3 Reference table for interpretation of result TSI

(Bergey s Manual, 9th edition; web link)

REACTION EXPLANATION
Acid butt(yellow), alkaline slant (red) Glucose fermented
Acid throughout medium, butt and slant yellow Lactose or sucrose or both fermented
Gas bubbles in butt, medium sometimes split Aerogenic culture
Blackening in the butt Hydrogen Sulfide produced
Alkaline slant and butt (medium entirely red) None of the three sugars fermented

Table.4 TSI result

Isolate number Slant Butt Gas Result

1 Yellow Yellow No gas Lactose or sucrose or both
production fermented
2 Red Yellow No gas Glucose fermented
production
Control Yellow Yellow Gas production Lactose or sucrose or both
(E.coli) fermented and Aerogenic
culture.

Table.5 Effect of chromium on growth of C2

Time (mins) CFU/ml

10 ³ 10
10 Lawn growth 56
20 Lawn growth 17
30 Lawn Growth 15
40 - -
50 - -
60 - -

Photo.1 Glowing Bioluminescent bacterial isolated colonies of C1 and C2 on LA

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Graph.1 Grwoth curve of Isolate C1

Graph.2 Growth curve of Isolate C2

Taken efforts were finally lead to successful pollutant toxicity testing (Engebrech et al.,
identification of bioluminescent bacteria; 1983; Thomulka and Peck, 1995; Kuruvet et
two well isolated colonies of bioluminescent al., 2005; Charrier et al., 2010; Kumar,
bacteria from gut flora of Indian Mackerel 2010, Gokhale et al., 2012). This study
on luminous agar were identified as Vibrio helped to perform the TVC of the isolate C2
(Bergey s Manual, 9th edition). Growth on
TCBS agar confirmed that the
bioluminescent isolate belongs to the genus at specific concentration of 10 ³ and 10 in
Vibrio. Growth on TCBS agar also state that
one of the isolate C2 may be a late sucrose
fermenter as we know there are some presence of pollutant chromium of
species that shows slow growth and also concentration 1ppm (APHA, 2012;
ferment sucrose slowly as they showed late Tchobanoglous, 2004). Chromium is
green coloured colonies(Musa et al., 2008; considered nonessential for plants, but an
Ransanga et al., 2012). The growth curve of essential trace element in animals.
the isolates gave the idea about the log phase Hexavalent compounds have been shown
of the organism. In this phase the carcinogenic by inhalation and are corrosive
bioluminescent intensity is prominent to tissue. The chromium guidelines for
(Engebrech et al., 1983; Kumar, 2010, natural water are linked to the hardness or
Gokale et al., 2012). As mentioned above alkalinity of the water (i.e., the softer the
isolate C2 showed steady growth in log water, the lower the permitted level for
phase i.e. steady intensity of light; so we chromium). The United Nations Food and
prefer isolate C2 for further examination in Agriculture Organization recommended

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 Int.J.Curr.Microbiol.App.Sci (2015) 4(10): 23-32

maximum level for irrigation waters in Berleman, J. 2009. Analysis of inter-species

100 g/L (APHA, 2012; Tchobanoglous, interaction and metabolite excretion in
2004). This minute amount also affects the bioluminescent marine bacteria isolates.
bacterial growth as the results showed MBC Microbial diversity. MA.
declining number of colonies of organisms; University of Iowa, IA.
we may say that the toxicity testing of Boynton, L. 2009. Using bioluminescent
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